In April 1993, tuberculosis was declared a global health emergency--the first such designation in the history of the World Health Organization. The distinction is regrettably justified because tuberculosis remains one of the largest causes of disease and death in the world (37), due in part to the increased susceptibility of HIV infected individuals and the ominous emergence of multi-drug resistant strains in both industrialized and developing countries. Effective new tuberculosis control and prevention strategies will require additional knowledge of the causative agent and its interaction with the human host.
In this regard, the determination of the genomic sequence of Mycobacterium tuberculosis (7) has provided many new opportunities for studying tuberculosis pathogenesis. Many of the genes in the genome of M. tuberculosis have no known function. A first step in establishing a function of an unknown gene lies in the generation of a mutation in that gene and characterization of the resulting mutant strain. Studying the behavior of these mutants in model systems of tuberculosis could reveal the mechanisms by which M. tuberculosis multiplies within the host cells and resists the immune effector functions of the host.
The availability of the M. tuberculosis genome sequences and the development of successful transformation protocols for the slow growing mycobacteria (32, 34) make the engineering of specific mutations readily achievable, but the introduction of these mutated alleles into their homologous sites in the chromosome, i.e. allelic exchange, has been notoriously difficult in this organism. Slow growing mycobacteria such as M. bovis BCG and M. tuberculosis can integrate exogenous DNA into their chromosome by both illegitimate and homologous recombination (13, 16). In recent years, numerous successful gene disruptions in various mycobacterial species were reported by using short (1, 13, 28) or long linear DNA fragments (2) as homologous DNA substrates. A "suicidal" vector approach, using recombinant plasmids unable to replicate in mycobacteria, was also extensively used to achieve allelic exchange in both fast- and slow-growing mycobacteria (5, 14) (22) (23) (24, 25, 31). Unfortunately, it is very difficult to estimate the real frequency of the allelic exchange events in these experiments due to the low number of transformants obtained, especially when using slow-growing mycobacteria. This led to the general conclusion that homologous recombination in the slow-growing mycobacteria is inefficient (16).
A two-step selection method using a selectable and counter-selectable marker positioned on either replicating or non-replicating plasmids has been successfully used in M. smegmatis (14, 24). Further, use of a conditionally replicating temperature sensitive plasmid as a delivery vector has greatly improved reproducibility of the allelic exchange in the slow growing mycobacteria (23).
However, the natural mechanisms of exchange of genetic information, such as conjugation or transduction, would be an alternative strategy to introduce homologous DNA into mycobacteria with high efficiency. Although conjugation has been described for M. smegmatis (17, 20, 33), it has not been demonstrated in the slow-growing mycobacteria. Similarly, transduction has been reported for M. smegmatis (26), but not for BCG or M. tuberculosis.
Because the creation of mutants in M. tuberculosis and BCG is of essential importance in the analysis of gene function, it is desirable to develop effective means and methods for allelic exchange for M. tuberculosis, BCG and other slow-growing mycobacteria. Methods for efficient allelic exchange would facilitate the definition of wildtype and mutant genes of M. tuberculosis and BCG mycobacteria, and thereby provide the necessary tools for understanding the mechanisms by which these mycobacteria survive and replicate. In addition, it would further the development of vaccines and new drugs effective in the treatment of infection caused by M. tuberculosis, BCG, and other mycobacteria.